Pyrrolidine derivatives for use in treating heaptitis c virus infection

ABSTRACT

The present invention relates to novel hepatitis C virus (“HCV”) protease inhibitors or other flavivirus protease inhibitors having the general structure (I) or (II), pharmaceutical compositions containing one or more such inhibitors, methods for preparing such inhibitors, uses of these compounds to treat hepatitis C and related disorders together with their use for their activity towards NS3 serine protease, intermediary compounds for the method of preparation of said compounds and screening methods. The invention specifically discloses novel chemical compounds as inhibitors of the HCV serine protease.

The present invention relates to novel hepatitis C virus (“HCV”) NS3serine protease inhibitors or other flavivirus protease inhibitors,pharmaceutical compositions containing one or more such inhibitors,methods for preparing such inhibitors, uses of these compounds to treathepatitis C and related disorders together with their use for theiractivity towards NS3 protease, intermediary compounds for the method ofpreparation of said compounds and screening methods. The inventionspecifically discloses novel chemical compounds as inhibitors of the NS3protease.

SCIENTIFIC BACKGROUND

Hepatitis C virus (HCV) is a (+)-sense single-stranded RNA virus thathas been implicated as the major causative agent in non-A, non-Bhepatitis (NANBH), particularly in blood-associated NANBH (BB-NANBH)(see, International Patent Application Publication No. WO 89/04669 andEuropean Patent Application Publication No. EP 381 216).

HCV has been implicated in cirrhosis of the liver and in induction ofhepatocellular carcinoma. The prognosis for patients suffering from HCVinfection is currently poor. HCV infection is more difficult to treatthan other forms of hepatitis due to the lack of immunity or remissionassociated with HCV infection.

Current data indicates a less than 50% survival rate at four years postcirrhosis diagnosis. Patients diagnosed with localized resectablehepatocellular carcinoma have a five-year survival rate of 10-30%,whereas those with localized unresectable hepatocellular carcinoma havea five-year survival rate of less than 1%.

Hepatitis C Infection

Hepatitis C has emerged in recent years as a common cause of liverdisease with an estimated 170-million infected people worldwide.Hepatitis C virus (HCV) infection is characterized by viral persistenceand chronic liver disease in approximately 80% of the reported cases.Complications of chronic hepatitis C include cirrhosis in 20% of cases,as well as hepatocellular carcinoma, the incidence of which reaches 4%to 5% per year in patients with cirrhosis. Hepatitis C-related end-stageliver disease is now the principal indication for liver transplantationin industrialized countries (1).

Brief Summary on HCV Biology

HCV is a single-strand positive-sense RNA virus which belongs to theFlaviviridae family including also yellow fever, virus West Nile Fevervirus and dengue virus. Upon translation of its unique open readingframe, a single polyprotein or polypeptide is synthesized. Thispolypeptide is then cleaved by both host and viral proteases to formstructural proteins (2).

As this time, the mechanisms of HCV replication in infected cells remainpoorly known. It is thought that the RNA-dependent RNA polymerase(RdRp), along with other non structural proteins, and the HCV RNAtemplate and host cell factors, form a ribonucleoproteinic complex ofreplication wherein perinuclear membranous structures are alsoassociated. Said structures appear to be the site of HCV RNA replication(3-4). By analogy with other Flaviviridae, the strategy for replicationwithin this complex seems to be as follows. A (−) strand copy of the RNAgenome is produced, serving in turn as a template for the production ofa (+) strand RNA. Indeed, said (−) strand HCV RNA was detected invarious cells or tissues harboring HCV replication (5-7).

The HCV RdRp, like other viral RNA polymerases, has a high error rate,with misincorporation frequencies averaging about 10⁻⁴ to 10⁻⁵ per basesite in the absence of any proofreading mechanism. Consequently,mutations continuously accumulate in newly-generated HCV genomes duringreplication. Most mutant viral particles are replication deficient, butsome can propagate efficiently. The fittest infectious particles areselected continuously on the basis of their replication capacities andunder the selective pressure resulting from the environment, saidpressure being mainly generated by the immune response of infectedpatients. This accounts for the presence, in each infected individual,of a pool of genetically-distinct but closely-related HCV variants,collectively referred to as a “quasispecies” (8, 9).

The HCV non structural (NS) proteins are presumed to provide theessential catalytic machinery for viral replication. The NS proteins arederived by proteolytic cleavage of the polyprotein. NS3 is anapproximately 68 kDa protein, encoded by approximately 1893 nucleotidesof the HCV genome, and has two distinct domains: (a) a serine proteasedomain consisting of approximately 200 of the N-terminal amino acids;and (b) an RNA-dependent ATPase domain at the C-terminus of the protein.The NS3 protease is considered as a member of the chymotrypsin familybecause of similarities in protein sequence, overall three-dimensionalstructure and mechanism of catalysis. Other chymotrypsin-like enzymesare elastase, factor Xa, thrombin, trypsin, plasmin, urokinase, tPA andPSA. The HCV NS protein 3 (NS3) contains a serine protease activity thathelps process the majority of the viral enzymes, and is thus consideredessential for viral replication and infectivity. It is known thatmutations in the yellow fever virus NS3 protease decreases viralinfectivity. The first 181 amino acids of NS3 (residues 1027-1207 of theviral polyprotein) have been shown to contain the serine protease domainof NS3 that processes all four downstream sites of the HCV polyprotein.The HCV NS3 serine protease and other associated cofactor, NS4A or NS4B,helps process all of the viral enzymes, and is thus considered essentialfor viral replication. The HCV NS3 serine protease is responsible forproteolysis of the polypeptide (polyprotein) at the NS3/NS4a, NS4a/NS4b,NS4b/NS5a and NS5a/NS5b junctions and is thus responsible for generatingfour viral proteins during viral replication. Recently, it is has beenrevealed that NS3, NS4B and NS5B can interact to form a regulatorycomplex that could feature in the process of HCV replication (13).

This has made the HCV NS3 serine protease an attractive target forantiviral chemotherapy.

Hepatitis C Current Treatments and the Need for Future TherapeuticDevelopments

Treatment of chronic hepatitis C is currently based on the use ofrecombinant interferon-α (INF-α). INF-α can be administered three timesper week subcutaneously, or, when pegylated, once weekly. The antiviraleffects of the various forms of IFN-α are enhanced by the addition ofribavirin, a nucleoside analog, the mechanism of action of which remainsunclear (1, 10, 11). Currently, the combination of pegylated IFN-α andribavirin for 24 to 48 weeks leads to definitive viral clearance in 52%to 57% of cases, whereas the remaining patients keep ongoing replicationand remain exposed to disease development. These therapies suffer from alow sustained response rate and frequent side effects.

Moreover, no vaccine is available for HCV infection.

Hence, it is generally acknowledged that there is a strong and urgentneed for new and highly active anti-HCV molecules. In this respect, theviral protease such the NS3A protease represent putative interestingtargets.

WO 02/08244 describes compounds which are deemed to be active forinhibiting HCV NS3 serine protease activity.

The Need for Experimental Models for HCV Studies

HCV exhibits a rather strict species specificity, the infection capacityof which being restricted to humans. At this time, there is no reliableanimal model, except for the experimentally-infected Chimpanzee andanother primate called Tupaia. Up to recently, there was neitherreliable cell culture model for in vitro studies of HCV virus. This lackof experimental models has considerably hampered the research onbiological properties of HCV, as well as on new therapeutic solutionsfor HCV infection treatment.

A new long-term, culture system was recently developed (12). Accordingto this in vitro model system, human hepatocytes can be cultured formore than one month with a full maintenance of highly differentiatedliver functions including: 1) production of plasma proteins such asalbumine, alpha-1 antitrypsin, fibrinogen, coagulation factors; 2)production of urea; 3) production of apolipoproteines such as ApoA andApoB100; 4) response to cytokines including interleukin-1, interleukin-6and interferon; 5) expression and inducibility of cytochromes P450 genesuperfamily; 6) capacity to metabolize drugs and other xenobiotics; and7) expression and DNA-binding activity of liver-enriched transcriptionfactors such as C/EBP family. Thus, this in vitro model appears to beuseful as a system for investigatirig HCV infection (for furtherdetails, see reference 12 and example 18).

There is a need for new treatments and therapies for HCV infection andrelated infections. It is therefore an object of the present inventionto provide chemical compounds useful in the treatment or prevention oramelioration of one or more symptoms of hepatitis C.

The inventors have now discovered that some new compounds might modulatethe activity of NS3 serine protease.

SUMMARY OF THE INVENTION

The invention provides a novel class of chemical compounds. The same areuseful as inhibitors of serine protease, in particular inhibitors of theHCV NS3 serine protease, it is a further object to provide compositionscontaining at least one of these compounds useful for the treatment orprevention or amelioration of diseases related or due to an infection bya virus (flavivirus, such as Deng virus, Yellow fever virus, West Nilefever virus and HCV), bacteria or pathogen dependent upon a serineprotease for proliferation. A particular embodiment is to providecompositions containing at least one of these compounds useful for thetreatment or prevention or amelioration of one or more symptoms ofhepatitis C. Another object is to provide methods of preparingpharmaceutical formulations comprising at least one of these compounds.It is a further object to provide methods for preparing the compounds.It is a further object to provide the use of these compounds for thepreparation of compositions useful in the treatment, the prevention ofthe amelioration of diseases related or due to an infection by a virus(flavivirus, such as Deng virus, Yellow fever virus, West Nile fevervirus and HCV), bacteria or pathogen dependent upon a serine proteasefor proliferation. These composition are in particular useful in thetreatment, the prevention of the amelioration of one or more of thesymptoms of hepatitis C. The invention also provides methods ofevaluating the modulation properties of compounds towards NS3 protease.

A further object of the invention is to provide methods for modulatingthe activity of HCV, particularly of the HCV NS3 protease, using thecompounds of the invention.

An other object herein is to provide in vitro model useful forinvestigating HCV infection and particularly the process of HCV NS3protease.

Among the chemical compounds of the invention, compounds that inhibitHCV NS3 serine protease are preferred.

The present invention discloses compounds having the general structure(I) or (II) as follows:

wherein:

R1 represents the side chain of an amino acid or an amino acidderivative, preferably of hydrophobic nature, an alkyl, alkenyl, oralkynyl group having from 1 to 10 carbon atoms, including CH2CH3 andCH2CF3;

R2, identical or different, represents a hydrogen atom, an alkyl grouphaving from 1 to 10 carbon atoms, a hydroxyl function, an alkoxy group,or an (C2-14) aryloxy group, —R2 may also represent a carbonyl group(═O);

R3, identical or different, represents the side chain of an amino acidor an amino acid derivative, preferably of hydrophobic nature, an alkyl,alkenyl, or alkynyl group having from 1 to 10 carbon atoms, or a,substituted or not, (C2-14) aryl or (C2-14) aralkyl group, the arylmoiety thereof being optionally interrupted by at least one heteroatom;

R4 represents a hydrogen atom, an alkyl, alkenyl, or alkynyl grouphaving from 1 to 10 carbon atoms;

R5 represents a protecting group for the amine function;

R6 and R7 are the same or different and each represents a hydrogen atomor an, linear, branched, or cyclic, alkyl, alkenyl, or alkynyl grouphaving from 1 to 10 carbon atoms or a, substituted or not, (C2-14)arylor (C2-14)aralkyl group, the aryl moiety thereof being optionallyinterrupted with at least one heteroatom;

R8 and R9 are the same or different and each represents a hydrogen atomor an, linear, branched, or cyclic, alkyl, alkenyl, or alkynyl grouphaving from 1 to 10 carbon atoms or a, substituted or not, (C2-14) arylor (C2-14) aralkyl group, the aryl moiety thereof being optionallyinterrupted with at least one heteroatom;

R10 represents an aldehyde (—CHO), an acid group (≦COOH), a sulfonicacid (—SO2OH), —COCOOH group, a radical selected in the group consistingof: —COR, —COOR, —CONRR′, —COCOOR, —SO2NRR′ (a sulfonamide group),—CONHCOR, —COCONRR′, —CONHSO2R, —CHOHCOR, —CHOHCOOR, —CHOHCON—RR′, R andR′, identical or different, represent an hydrogen atom, a hydroxylradical, a linear, branched or cyclic alkyl, alkene or alkyne grouphaving from 1 to 10 carbon atoms, an alkoxy group, an amine group or a,substituted or not, (C2-14) aryl, (C2-14) aralkyl, or (C2-14) aralkoxygroup, the aryl moiety thereof being optionally interrupted with atleast one heteroatom;

n is 1 or 2;

their tautomers, optical and geometrical isomers, racemates, salts,hydrates and mixtures thereof.

According to a specific embodiment, the compounds of the presentinvention have the following formula (III):

wherein:

R1, R2, R4, R5, R6, R7, R8, R9, R10 and n are as defined above and R11represents a hydrogen atom, an alkyl group having from 1 to 10 carbonatoms inclusive or a carboxy protecting group;

their tautomers, optical and geometrical isomers, racemates, salts,hydrates and mixtures thereof.

In particular, the compounds of the present invention present theformula (III) as identified above, wherein:

R1 represents an alkyl group having from 1 to 10 carbon atoms inclusiveor the side chain of an amino acid or an amino acid derivative,including CH2—CH3 and CH2CF3;

R2 represents a hydroxyl group, an alkoxy group having from 1 to 10carbon atoms, or —R2 may also represent a carbonyl group (═O);

R4 represents a hydrogen atom;

R5 represents an amine protecting group;

R6 and R7 are the same or different and each represents a hydrogen atom,a linear or branched alkyl group having from 1 to 10 carbon atoms or acycloalkyl group having from 1 to 10 carbon atoms, including acyclohexyl derivative;

R8 and R9 are the same or different and each represents a hydrogen atomor a linear or branched alkyl group having from 1 to 10 carbon atomsinclusive;

R10 represents an acid group, an ester group, an alkanoyl group, aketo-acid, a keto-ester, a keto-amide or a α-hydroxy-keto derivative;

R11 represents a hydrogen atom, an alkyl group having from 1 to 10carbon atoms inclusive or a carboxy protecting group; and

n is 1 or 2;

their tautomers, optical and geometrical isomers, racemates, salts,hydrates and mixtures thereof.

The compounds of the present invention may have one or more asymmetriccenters and it is intended that stereoisomers (optical isomers), asseparated, pure or partially purified stereoisomers or racemic mixturesthereof are included in the scope of the invention.

In particular, the preferred compounds are the following isomers:

Within the context of the present application, the terms alkyl, alone orin combination with any other term, denote linear, branched or cyclic,groups containing from 1 to 10 carbon atoms, preferably from 1 to 6carbon atoms. An alkanoyl group is a —CO-alkyl group, the alkyl groupbeing as defined above. An alkoxy group is a —O-alkyl group, the alkylgroup being as defined above.

The alkyl groups may be linear or branched. Examples of alkyl groupshaving from 1 to 10 carbon atoms inclusive are methyl, ethyl, propyl,isopropyl, t-butyl, isobutyl, n-butyl, pentyl, isopentyl, hexyl, heptyl,octyl, nonyl, decyl, 2-ethylhexyl, 2-methylbutyl, 2-methylpentyl,1-methylhexyl, 3-methylheptyl and the other isomeric forms thereof.Preferably, the alkyl groups have from 1 to 6 carbon atoms.

The unsaturated alkyl groups having from 2 to 10 carbon atoms includethe alkenyl groups. Among the alkenyl groups, a radical containing from2 to 10 carbon atoms; particularly 2 to 6 carbon atoms, and having oneor more carbon-to-carbon double bonds, such as ethenyl, 1-propenyl,2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl, 2-pentenyl,3-pentenyl, 4-pentenyl, 1 -hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl,5-hexenyl and the isomeric forms thereof. The allyl radical, ispreferred.

The unsaturated alkyl groups having from 2 to 10 carbon atoms includethe alkynyl groups. Among the alkynyl groups, a radical containing from2 to 10 carbon atoms; particularly 2 to 6 carbon atoms, and having oneor more carbon-to-carbon triple bonds, such as ethynyl, 1-propynyl,2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl,3-pentynyl, 4-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, or5-hexynyl.

The alkyl, alkenyl or alkynyl groups may also be cyclic. This term,alone or in combination with any other term, refers to a stablenon-aromatic 3- to 8-membered carbon ring radical which is saturated orunsaturated and which may be optionally fused, for example benzofused,with one to three other ring, aryl, heterocyclic or heteroaryl rings.The cycloalkyl may be attached at any endocyclic carbon atom whichresults in a stable structure. Preferred carbocycles have 5 to 6carbons. Examples of carbocycle radicals include, but are not limitedto, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,indane, tetrahydronaphthalene and the like.

The term “protecting” refers to when the designated functional group isattached to a suitable chemical group (protecting group).

Examples of suitable amino protecting groups are described in Greene andWuts, Protective Group in Organic Synthesis, 3^(rd) edition, John Wiley& Sons, New York 1991 and are exemplified in certain of the specificcompounds used in this invention. The preferred amino protecting groupforms, with the nitrogen atom it is attached thereto, a urethane, amidegroup, or a sulfonamide derivative. It includes the allyloxycarbonyl(Alloc), benzyloxycarbonyl (Cbz), chlorobenzyloxycarbonyl,t-butyloxycarbonyl (Boc), fluorenylmethoxycarbonyl (Fmoc),isonicotinyloxycarbonyl (I-Noc) and any oxycarbonyl group (presenting analkyl group or aryl group having from 5 to 10 carbon atoms andoptionally heteroatoms). It includes also benzoyl, acetyl and any othercarbonyl group (presenting an alkyl group or aryl group having from 5 to10 carbon atoms and optionally heteroatoms); p-toluenesulfonyl,phenylsulfonyl, methanesulfonyl, trifluoromethanesulfonyl and any othersulfonyl group (presenting an alkyl group or aryl group having from 5 to10 carbon atoms and optionally heteroatoms).

The carboxy protective groups are known in the art (e.g Greene and Wuts,Protective Group in Organic Synthesis) and include aryl (such as thebenzyl group :Bn), allyl, or alkyl group.

The amino acid side chain includes any side chain of the naturallyoccurring (L form) or synthesized (L or D form) aminoacids (inparticular alpha-aminoacids and aminocyclopropanoic acid), or derivativethereof. It includes substituted side chain of amino acid. Thesubstituent can be for instance an halogen atom (Cl, Br, I, or F), analkyl, alkenyl, alkynyl group, a perhalogenoalkyl group (perfluoroalkylgroup) or an aromatic group. More particularly, said side chain mayrepresent —CH3, —CH(CH3)2, —CH2—CH(CH3)2, —CH(CH3)C2H5, H, —CH2OH,—CH2CH3, —CH(OH)CH3, —CH2SH, —CH2CF3, —CH2CH2CF3, —CH3C2H5,—(CH2)2—S—CH3, —CH2C6H5, —CH2—C6H4(OH), —CH2CONH2, —(CH2)2CONH2,—CH2COOH, —(CH2)2COOH, —(CH2)4NH2, —(CH2)3NHC(NH2)2, C6H5, etc.Preferably, the amino acid side chain is —CH2—CH(CH3)2 ; —CH3,—CH2CH3,—CH(CH3)2, —CH2CF3, —CH2CH2CF3, —CH3C2H5, C6H5, or —CH2CH═CH2.

The term aryl includes any aromatic group comprising preferably from 2to 14 carbon atoms, preferably from 6 to 14 carbon atoms, optionallyinterrupted by one or several heteroatoms selected from N, O, S or P.Most preferred aryl groups are mono- or bi-cyclic and comprises from 2to 14 carbon atoms, such as phenyl, α-naphtyl, β-naphtyl, antracenyl,fluorenyl, pyrrole, thiophene, thiazole, triazole, furane, or oxazolegroup.

The term aralkyl group generally stands for an aryl group attached to analkyl group as defined above, such as benzyl or phenylethyl.

The term aryloxy or aralkoxy group generally stands for an aryl oraralkyl group as defined above attached to an oxygen atom, such asphenoxy or benzyloxy, respectively.

Any of these groups or derivatives, including the amino acid side chain,may be optionally substituted with one or more groups selected fromhydroxyl group, halogen atom (including F, Cl or Br), cyano group, nitrogroup, acid group, ester (—COO(C1-C6)alkyl group), —OCO(C1-C6)alkylgroup, amide (—NHCO(C1-C6)alkyl or —CONH(C1-C6)alkyl group), sulfamide(—NHSO2(C1-C6) or —SO2—NH(C1-C6)alkyl group), alkyl, alkenyl, alkynyl,aryl, aralkyl group, (C1-C10)alkoxy radical, mono- or poly-cyclichydrocarbon group, C═O group, an amino group or a trifluoro(C1-C6)alkylgroup. These above groups are as defined above.

The term mono- or poly-cyclic hydrocarbon group is understood to referto hydrocarbon cyclic group having from 1 to 20 carbon atoms, preferablyfrom 3 to 20, and optionally interrupted with one or more heteroatomsselected in the group consisting of N, O, S and P. Among such mono- orpoly-cyclic hydrocarbon groups, cyclopropyl, cyclopentyl, cyclohexyl,cycloheptyl, 1- or 2-adamantyl groups, pyran, piperidine, pyrrolidine,morpholine, dioxan, tetrahydrothiophene, and tetrahydrofuran can becited.

Preferably, the amine group is NH2, NHCH3, NHC2H5, or a benzylamine.

The trifluoro(C₁-C₆)alkyl group is preferably the trifluoromethyl group.

Preferred Embodiments

According to a preferred embodiment, the compounds correspond to thegeneral formula (I) or (II), wherein R10 represents an acid group(—COOH), an ester group (—COOR), an alkanoyl group (—COR), a keto-acid(—COCOOH), a keto-ester (—COCOOR), a keto-amide (—COCONHR) or aα-hydroxy-keto (—CHOHCOR) derivative, wherein R independently representsa hydrogen atom, a hydroxyl radical, an alkyl group, an alkoxy group oran amine group as defined above. Where R10 represents —COOR, —COCOOR,—COR or —COCONHR group, R is preferably an alkyl group (in particular amethyl or ethyl radical). Where R10 is —CHOHCOR, R is preferably ahydroxyl radical, an alkoxy group (in particular methoxy, ethoxy) or anaralkoxy (in particular benzyloxy), or an amine group (in particularNH2, NHCH3, NHC2H5 or a benzylamine).

According to preferred embodiments, the compounds according to theinvention correspond to general formula (I) or (II) wherein:

R5 stands for acetyl, benzyloxycarbonyl (Cbz) or t-butyloxycarbonyl(Boc) groups; and/or R1 stands for —CH₂—CH₃, —CH₂—CF₃, —CH₂—CH₂—CF₃,—CH₂CHCH₂ or —CH₂—CHMe₂; and/or R2 stands for t-butyloxy ; and/or R3stands for —(CH₂)₂COOH, —CH(CH₃)₂, or —(CH₂)₂COOCH₃; and/or R10 is acid,—CHOHCOR, with R is OH or an alkoxy group (preferably methoxy orethoxy), keto-acid, keto-ester (preferably —COCOOMe, —COCOOEt orCOCOOBn), keto-amide (preferably COCONHMe, COCONHEt or COCONHBn); and/orR4 is H; and/or R6 is H; and/or R7 is H; and/or R8 is H; and/or R9 is H;and/or R10 is H and/or n=1.

A particular preferred group of compounds according to the presentinvention, are compounds of formula (I).

Specific examples of compounds of formula (I), (II) or (III) which fallwithin the scope of the present invention include the followingcompounds:

The compounds of the present invention present the advantage to be nonbactericidal and/or non mutagenic. The ames test with four differentstrains, with or without metabolic activation, has been carried out withthe above cited compounds (in particular, compound 5). The resultsthereof have shown that the compounds above identified (in particular,compound 5) are non mutagenic.

As stated earlier, the invention includes also tautomers, rotamers,enantiomers and other stereoisomers of the inventive compounds. Thus, asone skilled in the art appreciates, some of the inventive compounds mayexist in suitable isomeric forms. Such variations are contemplated to bewithin the scope of the invention.

As used herein, the compounds of this invention, including the compoundsof formula (I) or (II), are defined to include pharmaceuticallyacceptable derivatives or prodrugs thereof. A “pharmaceuticallyacceptable derivative or prodrug” means any pharmaceutically acceptablesalt, ester, salt of an ester, carbonate, salt of carbonate (likemethyl, ethyl, isopropyl, benzyl or t-butyl carbonate derivatives) orother derivative of a compound of this invention which, uponadministration to a recipient, is capable of providing (directly orindirectly) a compound of this invention.

Accordingly, this invention also provides prodrugs of the compounds ofthis invention, which are derivatives that are designed to enhancebiological properties such as oral absorption, clearance, metabolism orcompartmental distribution. Such derivations are well known in the art.As the skilled practitioner realizes, the compounds of this inventionmay be modified by appending appropriate functionalities to enhanceselective biological properties. Such modifications are known in the artand include those which increase biological penetration into a givenbiological compartment (e.g., blood, lymphatic system, central nervoussystem), increase oral availability, increase solubility to allowadministration by injection, alter metabolism and alter rate ofexcretion.

Particularly favored derivatives and prodrugs are those that increasethe bioavailability of the compounds of this invention when suchcompounds are administered to a mammal (e.g., by allowing an orallyadministered compound to be more readily absorbed into the blood), havemore favorable clearance rates or metabolic profiles, or which enhancedelivery of the parent compound to a biological compartment (e.g., thebrain or lymphatic system) relative to the parent species. Preferredprodrugs include derivatives where a group which enhances aqueoussolubility or active transport through the gut membrane is appended tothe structure of formula (I).

Bioavailability refers to the rate and extent to which the active drugingredient or therapeutic moiety is absorbed into the systemiccirculation from an administered dosage form as compared to a standardor control.

The compounds of the present invention may include salts thereof. Whenthe compounds according to the invention are in the forms of salts, theyare preferably pharmaceutically acceptable salts. Such salts includepharmaceutically acceptable acid addition salts, pharmaceuticallyacceptable base addition salts, pharmaceutically acceptable metal salts,ammonium and alkylated ammonium salts. Acid addition salts include saltsof inorganic acids as well as organic acids. Representative examples ofsuitable inorganic acids include hydrochloric, hydrobromic, hydroiodic,phosphoric, sulfuric, nitric acids and the like. Representative examplesof suitable organic acids include formic, acetic, trichloroacetic,trifluoroacetic, propionic, benzoic, cinnamic, citric, fumaric,glycolic, lactic, maleic, malic, malonic, mandelic, oxalic, picric,pyruvic, salicylic, succinic, methanesulfonic, ethanesulfonic, tartaric,ascorbic, pamoic, bismethylene salicylic, ethanedisulfonic, gluconic,citraconic, aspartic, stearic, palmitic, EDTA, glycolic, p-aminobenzoic,glutamic, benzenesulfonic, p-toluenesulfonic acids, sulphates, nitrates,phosphates, perchlorates, borates, acetates, benzoates,hydroxynaphthoates, glycerophosphates, ketoglutarates and the like.Further examples of pharmaceutically acceptable inorganic or organicacid addition salts include the pharmaceutically acceptable salts listedin J. Pharm. Sci. 1977, 66, 2, which is incorporated herein byreference. Examples of metal salts include lithium, sodium, potassium,magnesium salts and the like. Examples of ammonium and alkylatedammonium salts include ammonium, methylammonium, dimethylammonium,trimethylammonium, ethylammonium, hydroxyethylammonium, diethylammonium,butylammonium, tetramethylammonium salts and the like. Examples oforganic bases include lysine, arginine, guanidine, diethanolamine,choline and the like.

The pharmaceutically acceptable salts are prepared by reacting thecompound of formula I or II with 1 to 4 equivalents of a base such assodium hydroxide, sodium methoxide, sodium hydride, potassiumt-butoxide, calcium hydroxide, magnesium hydroxide and the like, insolvents like ether, THF, methanol, t-butanol, dioxane, isopropanol,ethanol, etc. Mixture of solvents may be used. Organic bases likelysine, arginine, diethanolamine, choline, guanidine and theirderivatives etc. may also be used. Alternatively, acid addition saltswherever applicable are prepared by treatment with acids such ashydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid,phosphoric acid, p-toluenesulphonic acid, methanesulfonic acid, fonicacid, acetic acid, citric acid, maleic acid salicylic acid,hydroxynaphthoic acid, ascorbic acid, palmitic acid, succinic acid,benzoic acid, benzenesulfonic acid, tartaric acid and the like insolvents like ethyl acetate, ether, alcohols, acetone, THF, dioxane,etc. Mixture of solvents may also be used.

In another embodiment, this invention provides a pharmaceuticalcomposition comprising at least one compound as defined above. Thepharmaceutical composition generally additionally comprises apharmaceutically acceptable vehicle or carrier. Because of their HCVinhibitory activity; such pharmaceutical compositions possess utility intreating hepatitis C and related disorders.

In yet another embodiment, the present invention discloses methods forpreparing pharmaceutical compositions comprising the inventive compoundsas an active ingredient.

In the pharmaceutical compositions and methods of the present invention,the active ingredients will typically be administered in admixture withsuitable carrier materials suitably selected with respect to theintended form and administration, i.e. oral tablets, capsules (eithersolid-filled, semi-solid filled or liquid filled), powders forconstitution, oral gels, elixirs, dispersible granules, syrups,suspensions, and the like, and consistent with conventionalpharmaceutical practices.

The compounds may thus be formulated in various forms, including solidand liquid forms, such as tablets, capsules, gel, syrup, powder,aerosol, suppositories etc.

The compositions of this invention may contain physiologicallyacceptable diluents, fillers, lubricants, excipients, solvents, binders,stabilizers, and the like. Diluents that may be used in the compositionsinclude but are not limited to dicalcium phosphate, calcium sulphate,lactose, cellulose, kaolin, mannitol, sodium chloride, dry starch,powdered sugar and for prolonged release tablet-hydroxy propyl methylcellulose (HPMC). The binders that may be used in the compositionsinclude but are not limited to starch, gelatin and fillers such assucrose, glucose, dextrose and lactose.

Natural and synthetic gums that may be used in the compositions includebut are not limited to sodium alginate, ghatti gum, carboxymethylcellulose, methyl cellulose, polyvinyl pyrrolidone and veegum.Excipients that may be used in the compositions include but are notlimited to microcrystalline cellulose, calcium sulfate, dicalciumphosphate, starch, magnesium stearate, lactose, and sucrose. Stabilizersthat may be used include but are not limited to polysaccharides such asacacia, agar, alginic acid, guar gum and tragacanth, amphotsics such asgelatin and synthetic and semi-synthetic polymers such as carbomerresins, cellulose ethers and carboxymethyl chitin.

Solvents that may be used include but are not limited to Ringerssolution, water, distilled water, dimethyl sulfoxide to 50% in water,propylene glycol (neat or in water), phosphate buffered saline, balancedsalt solution, glycol and other conventional fluids.

Additionally, the compositions of the present invention may beformulated in a sustained release form to provide the rate controlledrelease of any one or more of the components or active ingredients tooptimize the therapeutic effects, i.e. HCV inhibitory activity and thelike. Suitable dosage forms for sustained release include layeredtablets containing layers of varying disintegration rates or controlledrelease polymeric matrices impregnated with the active components andshaped in tablet form or capsules containing such impregnated orencapsulated porous polymeric matrices.

Liquid form preparations include solutions, suspensions and emulsions.As an example may be mentioned water or water-propylene glycol solutionsfor parenteral injections or addition of sweeteners and pacifiers fororal solutions, suspensions and emulsions. Liquid form preparations mayalso include solutions for intranasal administration.

Aerosol preparations suitable for inhalation may include solutions andsolids in powder form, which may be in combination with apharmaceutically acceptable carrier such as inert gas, e.g. nitrogen.

For preparing suppositories, a low melting wax such as a mixture offatty acid glycerides such as cocoa butter is first melted, and theactive ingredient is dispersed homogeneously therein by stirring orsimilar mixing. The molten homogeneous mixture is then poured intoconvenient sized molds, allowed to cool and thereby solidify.

Also included are solid form preparations which are intended to beconverted, shortly before use, to liquid form preparations for eitheroral or parental administration. Such liquid forms include solutions,suspensions and emulsions.

The compounds of the invention may also be deliverable transdermally.The transdermal compositions may take the form of creams, lotions,aerosols and/or emulsions and can be included in the transdermal patchof the matrix or reservoir type as are conventional in the art for thispurpose.

Preferably, the compounds are administered orally, intravenously orsubcutaneously.

The dosages and dosage regimen in which the compounds of formula (I) areadministered will vary according to the dosage form, mode ofadministration, the condition being treated and particulars of thepatient being treated. Accordingly, optimal therapeutic concentrationswill be best determined at the time and place through routineexperimentation.

The compounds according to the invention can be used enterally orparenterally. Orally, the compounds according to the invention aresuitably administered at the rate of 100 μg to 100 mg per day per kg ofbody weight. The required dose can be administered in one or moreportions. For oral administration, suitable forms are, for example,tablets, gel, aerosols, pills, dragees, syrups, suspensions, emulsions,solutions, powders and granules. A preferred method of administrationconsists in using a suitable form containing from 1 mg to about 500 mgof active substance.

The compounds according to the invention can also be administeredparenterally in the form of solutions or suspensions for intravenous orintramuscular perfusions or injections. In that case, the compoundsaccording to the invention are generally administered at the rate ofabout 10 μg to 10 mg per day per kg of body weight; a preferred methodof administration consists of using solutions or suspensions containingapproximately from 0.01 mg to 1 mg of active substance per ml.

The compounds of the present invention can be used in a substantiallysimilar manner to other known anti-hepatitis C agents. For the compoundsof this invention, the anti-hepatitis C dose to be administered, whethera single dose, multiple dose, or a daily dose, will of course vary withthe particular compound employed because of the varying potency of thecompound, the chosen route of administration, the size of the recipient,and the nature of the patient's condition. The dosage to be administeredis not subject to definite bounds, but it will usually be an effectiveamount, or the equivalent on a molar basis of the pharmacologicallyactive free form produced from a dosage formulation upon the metabolicrelease of the active drug to achieve its desired pharmacological andphysiological effects. Someone skilled in the art of hepatitis Ctreatment will be able to ascertain, without undue experimentation,appropriate protocols for the effective administration of the compoundsof this present invention, such as by referring to the earlier publishedstudies on compounds found to have anti-hepatitis C properties.

Preferably, the pharmaceutical preparation is in a unit dosage form. Insuch form, the preparation is subdivided into suitable sized unit dosescontaining appropriate quantities of the active components, e.g., aneffective amount to achieve the desired purpose.

The quantity of the inventive active compound in a unit dose ofpreparation may be generally varied and adjusted from about 0.2milligrams to about 1000 milligrams, preferably from about 0.5 to about950 milligrams, more preferably from about 1.0 to about 500 milligrams,and typically from about 1 to 250 milligrams, according to theparticular application. The actual dosage employed may be varieddepending upon the patient's age, sex, weight and severity of thecondition being treated. Such techniques are well known to those skilledin the art.

Generally, the human oral dosage form containing the active ingredientscan be administered 1 or 2 times per day. The amount and frequency ofthe administration will be regulated according to the judgment of theattending clinician. A generally recommended daily dosage for oraladministration may range from about 1.0 milligram to about 1,000milligrams per day, in single or divided doses.

Conventional methods for preparing tablets are known. Such methodsinclude dry methods such as direct compression and compression producedby compaction, or wet methods or other special procedures. Conventionalmethods for making other forms for administration such as, for example,capsules, suppositories and the like are also well known.

The compounds of the invention can be used alone or in combination withimmunomodulatory agents, such as interferons, in particularα-interferon; other antiviral agents such as ribavirin and amantadine;other inhibitors of hepatitic C protease; inhibitors of other targets inthe HCV life cycle including the helicase, polymerase, metalloprotease,or internal ribosome entry; or combinations thereof. The other activeingredient can be administered in combination with a compound of thepresent invention either simultaneously, separately, or sequentially.

By “simultaneous”, it is meant that the two compounds are administeredat the same time, though not necessarily in the same composition. By“sequential”, it is meant that the two compounds are administered withina time period such that the first of the two compounds is still activein the patient when administration of the second of the two compoundsoccurs.

Preferably, “sequential” means within the same 24 hour, preferablywithin the same 12 hour, such as within the same 6, 3, 1, half orquarter hour time period.

The present invention provides compounds that are useful as serineprotease inhibitors, in particular as viruses (preferably flavivirus,such as dengue virus, Yellow fever virus, West Nile fever virus andHCV), bacteria or any other pathogens serine protease inhibitors, andmore preferably as HCV NS3 protease inhibitors. As such, they act byinterfering with the life cycle of the HCV virus and any other virus,bacteria or pathogen that are dependent upon a serine protease forproliferation and/or infection. Preferably, these compounds are usefulas antiviral agents.

Another embodiment of the invention discloses the use of thepharmaceutical compositions disclosed above for treatment of diseases,in particular for treating diseases related or due to an infection by avirus (preferably flavivirus, such as dengue virus, Yellow fever virus,West Nile fever virus and HCV), bacteria or pathogen dependent upon aserine protease for proliferation, more particularly for treating HCVinfection and the like. The method comprises administering atherapeutically effective amount of the inventive pharmaceuticalcomposition to a mammal (in particular a patient) having such a diseaseor diseases and in need of such a treatment.

The present invention relates to a method for the treatment of adisease, and in particular a disease related or due to an infection by avirus (preferably flavivirus, such as dengue virus, yellow fever virus,West Nile fever virus and HCV), bacteria or pathogen dependent upon aserine protease for proliferation. More particularly, the presentinvention relates to a method to treat HCV infection and the like, suchas the complications of hepatitis C, in particular chronic hepatitis,cirrhosis, hepatocellular carcinoma, or extra hepatic manifestation,comprising administering to a mammal (in particular a patient) in needof such treatment an effective amount of at least one compound of thepresent invention as defined herein.

A further object of this invention is the use of an effective amount ofat least one compound of formula (I) or (II) as defined above for thepreparation of pharmaceutical composition for the treatment of a diseaseassociated or due to an infection by a virus (preferably flavivirus,such as dengue virus, Yellow fever virus, West Nile fever virus andHCV), bacteria or pathogen dependent upon a serine protease forproliferation. More particularly, the pharmaceutical composition isintended to treat a disease associated with HCV infection.

Because of their anti-pathogen properties and particularly their HCVinhibitory activity, the compounds of this invention are suitable fortreating a variety of diseases in a variety of conditions. In thisregard, “treatment” or “treating” include both therapeutic andprophylactic treatments. Accordingly, the compounds may be used at veryearly stages of a disease, or before early onset, or after significantprogression.

Typical examples of diseases associated with HCV infection includechronic hepatitis, cirrhosis, hepatocarcinoma, or extra hepaticmanifestation for instance. The compounds of this invention areparticularly suited for the treatment of hepatitis C and complicationsthereof, in particular cirrhosis or hepatocellular carcinoma.

Another embodiment of the invention discloses a method of preparing thecompounds disclosed herein. The compounds may be prepared by severaltechniques known in the art. Representative illustrative procedures areoutlined in the following reaction schemes (FIGS. 1-4). It is to beunderstood that while the following illustrative schemes describe thepreparation of a few representative inventive compounds, suitablesubstitutions of any substituent will result in the formation of thedesired compounds based on such substitution. Such variations arecontemplated to be within the scope of the invention.

In particular, they can be prepared by the following general method(s):

Compounds of general formula (I) and (II) can be obtained by generalpeptide coupling methods of a central proline derivative with usual ormodified amino acids derivatives. The reactants used in these proceduresare standard peptide coupling reagents like BOP, PyBOP, DCC, HOBT, etc.The conditions are mainly in usual organic solvents, such asethylacetate (EtOAc), dichloromethane (DCM), dichloroethane (DCE),dimethylformamide (DMF) and usually at room temperature (18-25° C.).

General Strategy : A proline moiety including specific modifications wasfirstly prepared before to be C-terminal coupled to a modifiedamino-acid (including ketone, ketoacid, ketoamide, or ketoesterderivatives). Following the standard N-deprotection of the resultingcompound, the last synthon (N-protected amino acid derivative) wasfinally added to provide an intermediate compound which finally providesafter standard deprotection steps the target molecule (compound of thepresent invention).

Following the above described methods for preparing the compounds offormula (I) or (II), the present invention further provides compoundscorresponding to the following formula (V):

wherein R2, R3, R4, R5, R6, R7, R8, and R9 are as defined above and R12represents a hydrogen atom, an alkyl group (in particular, methyl,tert-butyl, or ethyl), alkenyl (allyl), an aralkyl (for instance,benzyl) or a cycloalkyl group; and n is 1 or 2;

their tautomers, optical and geometrical isomers, racemates, salts,hydrates and mixtures thereof.

The above cited groups are as defined and specified for compounds offormulae (I) and (II). They can also be substituted as mentioned above.

In a particular embodiment, the present invention provides compounds offormula (V) as defined above, wherein R2 represents an alkoxy group, oran aryloxy group, R4 is an hydrogen atom, R6, R7, R8, and R9,independently from each other, represents a hydrogen atom, an alkyl, analkoxy group, a cycloalkyl, an acid group, an ester group, an alkanoylgroup, a keto-acid, a keto-ester, a keto-amide or a α-hydroxy-ketogroup; R5 represents an amine protecting group; R12 represents ahydrogen atom, an alkyl group or a cycloalkyl group; and n is 1 or 2,

The compounds of formula (V) may have one or more asymmetric centers andit is intended that stereoisomers (optical isomers), as separated, pureor partially purified stereoisomers or racemic mixtures thereof areincluded in the scope of the invention.

In particular, the preferred compounds of formula (V) are the followingisomers:

According to a specific embodiment, compounds of formula (V) correspondto compounds of formula (V) wherein R6, R7, R8 and R9, independentlyfrom each other, represents a hydrogen atom, an alkyl, an alkoxy group,or a cycloalkyl group, and preferably a hydrogen atom.

Specific examples of compounds of formula (V) which fall within thescope of the present invention include the following compound:

According to another specific embodiment, compounds of formula (V)correspond to compounds of formula (V) wherein at least one of R6, R7,R8 and R9 represents an acid group, an ester group, an alkanoyl group, aketo-acid, a keto-ester, a keto-amide or a α-hydroxy-keto group, asdefined above.

Compounds of formula (V) may be used either as intermediate compounds toprepare compounds of formula (I) or (II) or as active ingredients, suchas the compounds of formula (I) and (II) of the present invention and inparticular as antiviral agents, such as antiviral HCV agent.

NS3 Inhibitory Activity

Primary cultures of normal human hepatocytes infected in vitro andsupporting sustained HCV replication, the closest model to the in vivoHCV-infected human liver, were used to show that the compounds of theinvention are able to block HCV replication in infected humanhepatocytes. In view of these results, said compounds appear to beprimarily involved through their nonspecific antiviral effect. Theprimary cultures of human hepatocytes described herein provide a good invitro model for studying intrinsic HCV resistance to the antiviralproperties of these compounds, and for screening the potentialtherapeutic capacity of new molecules, e.g., the chemical compounds ofthe invention, to inhibit HCV infection and/or replication.

In an alternate embodiment, this invention provides a method ofevaluating the modulation properties of compounds towards NS3 serineprotease, particularly HCV NS3 serine protease, said methodsimplementing in vitro primary cultures of human hepatocytes describedherein. In this respect, the present invention provides a method forscreening and/or characterizing compounds that present antiviralactivity, in particular antiviral HCV activity, by implementing in vitroprimary cultures of human hepatocytes described herein. In particularsaid methods comprise:

-   -   a) contacting a test compound with the in vitro primary cultures        of human hepatocytes in presence of HCV or active part thereof,        and    -   b) determining the antiviral activity of the test compound in        comparison with the antiviral activity of one of the compounds        of the present invention.        The in vitro primary cultures of human hepatocytes is more        specifically described in reference 12, which is incorporated        herein by reference.

More particularly, the in vitro primary cultures of human hepatocyteswere prepared as follows: hepatocytes were prepared from liver tissueresected from donors who tested negative for HCV, and inoculation wasperformed 3 days after plating with 33 HCV serum samples of differentvirus load and genotype. The presence of intracellular HCV RNA, detectedby a strand-specific rTth RT-PCR assay, was used as evidence ofinfection. A kinetics analysis of HCV replication revealed thatintracellular negative-strand RNA appeared at day 1 post-infection witha maximum level at days 3 and 5, followed by a decrease until day 14. Atday 5, the copy level of viral RNA was estimated to be amplified atleast 15-fold in infected cells. The level of intracellular HCV RNA inresponse to different serum samples was reproducible from one hepatocyteculture to another, suggesting that there is no inter-individualvariability in the susceptibility of hepatocytes to HCV infection. Thesefindings indicated that adult human hepatocytes in primary cultureretain their susceptibility to in vitro HCV infection and support HCVRNA replication. This model represents therefore a valuable tool for thestudy of initial steps of the HCV replication cycle and for theevaluation of antiviral molecules.

In another embodiment, the invention discloses methods of decreasingprotease activity in a mammal comprising the step of administrating tosaid mammal any of the pharmaceutical compositions and combinationsdescribed above. If the pharmaceutical composition comprises only acompound of this invention as the active component, such methods mayadditionally comprise the step of administering to said mammal an agentselected from an immunomodulatory agent, an antiviral agent, a HCVprotease inhibitor, or an inhibitor of other targets in the HCV lifecycle. Such additional agent may be administered to the mammal prior to,concurrently with, or following the administration of the HCV inhibitorcomposition.

In a preferred embodiment, these methods are useful in decreasing HCVNS3 serine protease activity in a mammal. If the pharmaceuticalcomposition comprises only a compound of this invention as the activecomponent, such methods may additionally comprise the step ofadministering to said mammal an agent selected from an immunomodulatoryagent, an antiviral agent, a HCV protease inhibitor, or an inhibitor ofother targets in the HCV life cycle such as helicase, polymerase, ormetallo protease. Such additional agent may be administered to themammal prior to, concurrently with, or following the administration ofthe compositions of this invention.

In an alternate preferred embodiment, these methods are useful forinhibiting viral replication in a mammal. Such methods are useful intreating or preventing, for example, viral diseases, such as HCV. If thepharmaceutical composition comprises only a compound of this inventionas the active component, such methods may additionally comprise the stepof administering to said mammal an agent selected from animmunomodulatory agent, an antiviral agent, a HCV protease inhibitor, oran inhibitor of other targets in the HCV life cycle.

Such additional agent may be administered to the mammal prior to,concurrently with, or following the administration of the compositionaccording to this invention.

The compounds set forth herein may also be used as laboratory reagents.The compounds of this invention may be used to treat or prevent viralcontamination of materials and therefore reduce the risk of viralinfection of laboratory or medical personnel or patients who come incontact with such materials. These materials include, but are notlimited to, biological materials, such as blood, tissue, etc; surgicalinstruments and garments; laboratory instruments and garments; and bloodcollection apparatuses and materials.

Further aspects and advantages of this invention will be disclosed inthe following examples, which should be regarded as illustrative and notlimiting the scope of this application.

LEGEND TO THE FIGURES

FIG. 1: reaction scheme to prepare compound 5. The reactants and/orsolvents are as follows: a) Boc₂O, Dioxane, NaOH/H2O; b) BnOH, DCC/DMAP;c) TFA, DCE then BOP, DCE, fmoc-L-Hyp(OtBu), DIEA; d) DEA, CH3CN thenPyBOP, DIEA, Boc-L-Glu(OBn)OH; e) H2, Pd/C, EtOH

FIG. 2: reaction scheme to prepare intermediate compound 8. Thereactants and/or solvents are as follows: a) BnOH, DCM, DCC, DMAP; b)DEA, CH3CN then DIEA, DCM, PyBOP and Boc-L-Glu(OMe)-OH; c) H2, Pd/C,MeOH

FIG. 3: reaction scheme to prepare compounds 12 and 13. The reactantsand/or solvents are as follows: a) NC-CHPPh3, DCM, EDCl, DMAP; b) Pd/C,HCO2NH4, MeOH; c) O3, DCM, MeOH; d) H2, Pd/C; e) DIEA, PyBop, DCM; f)NaOH 1M, MeOH

FIG. 4: reaction scheme to prepare compounds 16 and 17. The reactantsand/or solvents are as follows: a) PyBOP, DIEA, DCM; b) O3, DCM, MeOH;c) NaOH 1M, MeOH

FIG. 5: Inhibitory effect of compound of example 5 expressed as theratio HCV RNA/GAPDH RNA

EXAMPLES

Preparation of the Compounds.

Nuclear magnetic resonance spectra were recorded on a Bruker WH-250 (250MHz) spectrometer. The chemical shift values are expressed in ppm (partper million) relative to tetramethylsilane as internal standard;s=singlet; d=doublet, dd=doublet of doublet; t=triplet; q=quartet;m=multiplet; bs=broad singlet. Coupling constants J are expressed in Hz.The relative integrals of peak areas agreed with those expected for theassigned structures. Thin layer chromatography (TLC) was performed onPOLYGRAM Sil G/UV 254 silica gel plates with fluorescent indicator andthe spots were visualized under 254 and 366 nm illumination. Proportionsof solvents used for TLC are by volume. All solvents and most chemicalswere purchased from Aldrich Chemical Co. and were used as received.Amino acids were purchased from Neosystem Group SNPE.

Abbreviations

Ac: Acetyl, Boc: t-butyloxycarbonyl, Boc₂O: di-t-butyl-dicarbonate; BOP:Benzotriazol-1-yloxyl-tris-(dimethylamino)-phosphoniumhexafluorophosphate; BnOH: Benzyl alcohol, CBD: Cellulose BindingDomain; DCC: N,N′-dicyclohexylcarbodiimide; DCE: 1,2-dichloroethane;DCM: dichloromethane; DEA: diethylamine; DIEA: diisopropylethylamine;DMAP: 4-dimethylaminopyridine; EDCI:1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; EGFP: Greenfluorescent protein; EtOAc: Ethyl Acetate; Fmoc:-(9H-fluoren-9-ylmethoxy)carbonyl; Gla: γ-Carboxyglutamic acid; Glu:glutamate, HOBT: 1-Hydroxybenzotriazole, Hyp: hydroxyproline; MeOH:Methanol; PMSF: Phenylmethylsulfonyl fluoride; PyBOP:Benzotriazol-1-yloxyl-tris-(pyrrolidino)-phosphoniumhexafluorophosphate; TFA: Trifluoro Acetic Acid, TPCK:L-1-4′-Tosylamino-2-phenylethyl chloromethyl ketone; Z:Benzyloxycarbonyl.

Example 1 Compound 1

A solution of 2-amino-4,4,4-trifluorobutyric acid (100 mg, 0.636 mmol)in dioxane (0.6 mL) and 1M NaOH_(aq) (1.2 mL) was stirred at 0° C. for10 min. Boc₂O (166 mg. 0.763 mmol) was then added and the mixture wasstirred at room temperature for 24 h. The pH was periodically adjustedto 9 by addition of a 1M NaOH_(aq) solution. The mixture was extractedwith Et₂O then acidified (pH=2) with the addition of 5% aqueous HClsolution. The acidic aqueous phase was extracted with EtOAc and combinedorganic phases were washed with brine. After drying on MgSO₄,concentration under reduced pressure afforded the protected amino acid 1(92 mg, 71%).

Example 2 Compound 2

Compound 1 (54 mg, 0.215 mmol) was suspended at room temperature in DCM(5 mL) with 0.06 mL of benzyl alcohol (0.537 mmol), DCC (44 mg, 0.236mmol) and DMAP (29 mg, 0.236 mmol). The solution was stirred for 18 h,filtered on Celite and successively washed with a 5% solution of aqueousHCl and a 10% solution of aqueous NaHCO₃. The organic phases were driedover MgSO₄, evaporated and the residue was purified by chromatography(eluent gradient hexane/ether 95:5 to 1:1) to give the pure compound 2(41 mg, 55%).

¹H NMR (CDCl₃) δ: 1.34 (s, 9H), 2.65 (m, 2H), 4.50 (m, 1H), 5.10 (s,2H), 5.18 (m, 1H), 7.30 (m, 5H).

Example 3 Compound 3

Compound 2 (664 mg, 1,91 mmol) was suspended in DCE at room temperatureand 10 eq of TFA (1,47 mmol) were added. The solution was stirred for 2h, evaporated and suspended in DCE (10 mL). Fmoc-L-Hyp(OtBu) (835 mg,2,0 mmol), 2 eq of DIEA (665 μL, 3,82 mmol) and 1,06 eq of BOP (897 mg,2,03 mmol) were added and the mixture was stirred for 20 h at roomtemperature. The solvent was then evaporated and the residue dissolvedin EtOAc. The organic phases was successively washed with aqueoussolutions of 5% citric acid and 5% NaHCO₃. The organic phases were driedover MgSO₄, evaporated and the residue was purified by chromatography(30% EtOAc/Hexane) to give the pure compound 3 (1,2 g, quantitative).This compound was directly used in the next step.

Example 4 Compound 4

Compound 3 (521 mg, 0.81 mmol) was treated with a solution of 10% DEA inCH₃CN and stirred for 20 h at room temperature. The mixture wasevaporated and dissolved in DMF (9 mL). Boc-L-Glu(OBn)OH (300 mg, 0.89mmol), 2 eq of DIEA (282 μL, 1.62 mmol) and 1.1 eq of BOP (394 mg, 0.89mmol) were then added and the mixture was stirred for 24 h. The solventwas evaporated and the residue dissolved in EtOAc. The organic phasiswas successively washed with aqueous solutions of 5% citric acid and 5%NaHCO₃ and finally dried over MgSO₄. The residue was purified bychromatography (30% EtOAc/Hexane) to give the pure compound 4 (72 mg,12%).

¹H NMR (CDCl₃) δ: 0.3 (s, 9H), 0.5 (s, 9H), 1.9-2.7 (m, 6H), 3.2 (m,1H), 3.5 (m, 1H), 4.0-4.5 (m, 3H), 4.9 (s, 4H), 5.0-5.2 (m, 1H), 7.2 (m,10H).

Example 5 Compound 5

Compound 4 (74 mg, 0.1 mmol) was dissolved in a mixture of ⅓ EtOAc/EtOHcontaining 20% Pd(OH)₂ (50 mg) and stirred under a hydrogen atmospherefor 12 h at room temperature. The mixture was then filtered on Celiteand evaporated. It was then dissolved in a minimum of Et₂O and few dropsof hexane were added. The precipitate was filtered and vacuum dried togive compound 5 (35 mg, 63%) as a white powder.

¹H NMR (CDCl₃) δ: 0.3 (s, 9H), 0.5 (s, 9H), 1.0-2.0 (m, 6H), 2.4-3.0 (m,2H), 3.2-3.8 (m, 3H), 4.6 (m, 1H), 5.0 (m, 1H), 6.0 (bs, 1H), 6.9 (bs,1H). SM (GT, FAB⁺): 556 (M+1H)⁺

Example 6 Compound 6

Fmoc-L-Hyp(OtBu) (410 mg, 1 mmol) was dissolved in DCM (15 mL). BnOH(1.5 mmol), DCC (1 mmol) and DMAP (0.1 mmol) were then added and stirredfor 18 h at room temperature. The precipitate was removed by filtrationthrough Celite and the filtrate was washed with 5% aqueous HCl solutionfollowed by saturated NaHCO₃. The organic phase was dried over MgSO₄,concentrated under reduced pressure and purified by flash chromatography(hexane/Et₂O from 95:5 to 1:1) to give compound 6 (300 mg, 60%).

¹H NMR (CDCl₃) δ: 1.36 (s, 9H), 2.30-2.45 (m, 2H), 3.55 (dd, 1H, J=5.0,10.0), 4.00 (m, 1H), 4.40-4.60 (m, 2H), 4.70 (m, 1H), 5.30 (d, 1H,J=2.2), 5.40 (d, 1H, J=4.8), 7.40-7.60 (m, 10H), 7.60-8.00 (m, 3H).

Example 7 Compound 7

Compound 6 (390 mg, 0.78 mmol) was dissolved in a solution of anhydrousCH₃CN (5 mL) with DEA (600 μL). The mixture was stirred for 3 h at roomtemprature. DCE (10 mL) was added and the mixture concentrated underreduced pressure. The residue was dissolved in DCE (5 mL) and DIEA (271μL, 1.56 mmol). Boc-L-Glu(OMe)OH.DCHA (380 mg, 0.858 mmol) and BOP (379mg, 0.858 mmol) were added. The mixture was allowed to stirr for 24 h atroom temperature. It was then washed with a 5% aqueous HCl solutionfollowed by a saturated aqueous NaHCO₃ and finally dried over MgSO₄.Concentration under reduced pressure gave a crude product which waspurified by flash chromatography (hexane/EtOAc 90:10 to 70:30) to give 7(294 mg, 72%).

¹H NMR (CDCl₃) δ: 1.1 (s, 9H), 1.4 (s, 9H), 1.5-1.8 (m, 2H), 2.0-2.2 (m,2H), 2.25-2.5 (m, 2H), 3.5 (m, 1H), 3.6 (s, 3H), 3.8 (s, 1H), 4.25 (m,1H), 4.4 (m, 1H), 4.6 (m, 1H), 5.1 (m, 2H), 5.3 (d, 1H), 7.2 (s, 5H).

¹³C NMR (CDCl₃) δ: 14.52, 21.31, 28.64, 37.24, 43.76, 51.91, 58.05,67.19, 69.80, 74.59, 79.88, 128.00, 128.89, 135.92, 155.74, 171.01,171.98, 173.68.

Example 8 Compound 8

Compound 7 (468 mg, 0.9 mmol) was dissolved in MeOH (25 mL) and 10% Pdon charcoal (400 mg) was added. The mixture was stirred at roomtemperature under a hydrogen atmosphere for 3 h. The catalyst wasremoved by filtration trough Celite and the filtrate concentrated. Thecrude acid 8 was immediately used without purification.

Example 9 Compound 9

Z-L-LeuOH (mg, 2.4 mmol) was dissolved in anhydrous DCM (24 mL). EDCI(464 mg, 2.42 mmol) and DMAP (29 mg, 0.242 mmol) were successively addedand the mixture was stirred for 30 min. PPh₃CHCN (729 mg, 2.42 mmol) wasthen added and the mixture was stirred at room temperature for 24 h.Reaction was quenched with 2% aqueous HCl solution. The organic phasewas then washed with saturated NaHCO3 aqueous solution, dried over MgSO₄and concentrated under reduced pressure. The crude solid was purified byflash chromatography (hexane/EtOAc 9:1 to 1:1) to give compound 9 (1.31mmol, 55%).

¹H NMR (CDCl₃) δ: 0.72 (d, 1H, J=6.0), 0.78 (d=3H, J=6.0), 0.89 (d, 3H,J=6.00), 1.60 (m, 2H), 4.68 (m, 1H), 4.90 (m, 1H), 5.50 (s, 2H),7.30-7.65 (m, 20H)

Example 10 Compound 10

In a flask connected to an oil bubbler, compound 9 (109 mg, 0.2 mmol)was dissolved in 5 mL of anhydrous DCM/MeOH (7:3). The solution was thencooled to −78° C. and ozone was bobbled through it. The gas evolutionwas stopped during 5 min every 10 min. The temperature was maintainedbetween −78° C. and −85° C. Despite the persistence of starting materialtraces, the reaction was stopped after 7 h by purging with nitrogen. Thesolution was stirred for 1 h at room temperature and concentrated underreduced pressure. The residue obtained was directly purified by flashchromatography (hexane/EtOAc 98:2 to 90:10) to give compound 10 (31 mg,50%).

¹H NMR (CDCl₃) δ: 0.75 (d, 3H, J=6.0), 0.85 (d, 3H, J=6.0), 1.35 (m,1H), 1.60 (m, 2H), 3.80 (s, 3H), 4.95 (m, 1H), 5.02 (s, 2H), 5.15 (s,1H), 7.23-7.32 (m, 5H).

¹³C NMR (CDCl₃) δ: 21.80, 23.70, 25.50, 40.60, 53.10, 56.40, 68.80,128.80, 129.20, 136.40, 156.30, 161.10, 168.22, 193.20.

Example 11 Compound 11

Compound 10 (31 mg, 0.1 mmol) was dissolved in MeOH (5 mL) containing10% Pd on charcoal (100 mg). The mixture was stirred for 24 h at roomtemperature under a hydrogen atmosphere. The catalyst was removed byfiltration through Celite and the filtrate concentrated. The crude amine11 was immediately used in the next step.

Example 12 Compound 12

To a solution of compound 8 (0.1 mmol) and 11 (0.1 mmol) in anhydrousDCE (2 mL) were added DIEA (0.035 mL, 0.2 mmol) and PyBOP (57 mg, 0.11mmol). The mixture was stirred for 24 h at room temperature and 5 mL ofwater were added. The organic phase was washed with 5% aqueous HClsolution followed by saturated NaHCO₃ before to be dried over MgSO₄.Filtration and concentration under reduced pressure gave a crude productwhich was purified by flash chromatography (hexane/EtOAc from 9:1 to7:3) to give compound 12 (0.02 mmol, 20%).

¹H NMR (CDCl₃) δ: 0.75-0.90 (m, 6H), 1.12 (s, 9H), 1.38 (s, 9H),1.40-1.65 (m, 3H), 1.65-2.10 (m, 3H), 2.10-2.45 (m, 4H), 3.62 (s, 3H),3.68 (m, 2H), 3.72 (s, 3H), 4.24 (m, 2H), 4.42 (m, 2H), 4.57 (m, 1H),5.30 (m, 1H), 6.76 (s, 1H) SM (GT, FAB⁺): 588 (M+1H)⁺

Example 13 Compound 13

Compound 12 (43 mg, 0.07 mmol) was dissolved in a mixture of MeOH (0.5mL) and 1N aqueous NaOH solution (0.15 mL). The mixture was stirred for1 h at room temperature, extracted with Et₂O and then acidified (pH=2)by the addition of 5% aqueous HCl solution. The acidic aqueous phase wasextracted with EtOAc and the combined organic phases washed with brineand dried over MgSO₄. The crude product was purified by precipitation(addition of hexane in a DCM solution) to give compound 13 (22 mg, 56%)as a white powder.

¹H NMR (CDCl₃) δ: 0.60-0.70 (m, 6H), 0.96 (s, 9H), 1.05 (m, 1H), 1.22(s, 9H), 1.20-1.55 (m, 3H), 1.60-2.04 (m, 4H), 2.13-2.30 (m, 2H), 3.34(m, 1H), 3.62 (m, 1H), 4.00-4.21 (m, 4H), 4.42 (m, 1H), 5.53 (m, 1H),6.96 (m, 1H) SM (GT, FAB⁺): 559 (M+1H)⁺

Example 14 Compound 14

Compound 9 (483 mg, 0.90 mmol) was dissolved in MeOH (30 mL) and 10% Pdon charcoal (1.4 g) was added carefully at room temperature followed byammonium formate (2.1 g). After 10 min carbon dioxide evolution wasobserved. When TLC indicated complete conversion (30 min), the catalystwas removed by filtration and washed thoroughly with EtOAc. Combinedorganic phases were washed with water followed by brine and dried overMgSO₄. Concentration under reduced pressure gave 14 which was usedwithout further purification.

Example 15 Compound 15

To a solution of compound 8 (0.9 mmol) and compound 14 (0.9 mmol) inanhydrous DCE (4 mL) were added DIEA (0.31 mL, 1.80 mmol) and PyBOP (515mg, 0.99 mmol). The mixture was stirred for 24 h at room temperature and10 mL of water were added. The organic phase was washed with 5% aqueousHCl solution followed by saturated NaHCO₃ before to be dried over MgSO₄.Filtration and concentration under reduced pressure gave a crude productwhich was purified by flash chromatography (hexane/EtOAc from 9:1 to1:1) to give compound 15 (0.48 mmol, 54%).

¹H NMR (CDCl₃) δ: 0.88 (d, 3H, J=5.9), 0.92 (d, 3H, J=5.9), 1.12 (s,9H), 1.30 (m, 1H), 1.33 (s, 9H), 1.50-2.00 (m, 7H), 2.30 (m,2H), 3.35(m, 1 H), 3.60 (s, 3H), 3.75 (m, 1H), 4.40 (m, 2H), 5.00 (m, 1H), 5.25(m, 1H).

Example 16 Compound 16

In a flask connected to an oil bubbler, compound 15 (185 mg, 0.22 mmol)was dissolved in 6 mL of anhydrous DCM/MeOH (7:3). The solution was thencooled to −78° C. and ozone was bubbled through it. The gas evolutionwas stopped during 5 min every 10 min. The temperature was maintainedbetween −78° C. and −85° C. Despite the persistence of starting materialtraces, the reaction was stopped after 7 h by purging with nitrogen. Thesolution was stirred for 1 h at room temperature and concentrated underreduced pressure. The residue obtained was directly purified by flashchromatography (hexane/EtOAc 98:2 to 90:10) to give compound 16 (82 mg,64%).

¹H NMR (CDCl₃) δ: 0.86 (d, 6H, J=5.9), 1.11 (s, 9H), 1.20 (t, 1H,J=5.9), 1.36 (s, 9H), 1.50-2.10 (m, 6H), 2.45 (m, 3H), 3.40 (m, 1H),3.61 (s, 3H), 3.70 (m, 1H), 3.80 (s, 3H), 4.35-4.45 (m, 2H), 4.60 (dd,1H, J=3.0, 8.5), 5.00 (m, 1H), 5.25 (d, 1H).

¹³C NMR (CDCl₃) δ: 192.07 (ketone).

SM (GT, FAB⁺): 586 (M+1H)⁺

Example 17 Compound 17

Compound 16 (50 mg, 0.08 mmol) was dissolved in a mixture of MeOH (1 mL)and 1N aqueous NaOH solution (0.33 mL). The mixture was stirred for 1 hat room temperature, extracted with Et₂O and then acidified (pH=2) bythe addition of 5% aqueous HCl solution. The acidic aqueous phase wasextracted with EtOAc and the combined organic phases washed with brineand dried over MgSO₄. The crude product was purified by precipitation(addition of hexane in a DCM solution) to give compound 17 (25 mg 52%)as a white powder.

¹H NMR (CDCl₃) δ 1.10 (s, 9H), tBu) and 1.35 (s, 9H, tBu); disappearanceof OMe peaks.

SM (GT, FAB⁺): 558 (M+1H)⁺

Example 18 Compounds 18 and 19

Solid Phase Synthesis Method:

Compound 18 and 19 were produced by solid-phase peptide synthesis, usingthe N-alpha-9-fluorenylmethyloxycarbonyl (Fmoc)/t-butyl strategy. Astepwise peptide chain assembly was achieved using an automated peptidesynthesizer (Applied Biosystems, model A433). In each case, peptideassembly was performed on 400 mg of Rink acid resin (100-200 mesh)substituted at 0.43 mmole reactive groups per gram of resin, using 1mmole of Fmoc-amino acid derivatives. The Fmoc-amino acid derivativeswere coupled (20 min) as their hydroxybenzotriazole active esters inN-methyl pyrrolidone (5.8-fold excess). After peptide chain assembly,the peptide resin was treated for 10 min at 25° C. with 5 ml of amixture of trifluoroacetic acid (TFA)/dichloromethane (10:90, v/v).After filtration of the mixture, the crude peptide was directlyrecovered in 50 ml H2O, instead of being classically precipitated bydiethyl oxide. The crude 1, or its derivatives, was freeze-dried andlyophilised. After lyophilisation, the peptide was solubilised in 4 mlH2O and purified to homogeneity by analytical C18-reversed phase highperformance liquid chromatography (C-18 chromolith column, 150×10 mm;60-min linear gradient of 0% to 60% buffer B (buffer B=0.08% (v/v)TFA/acetonitrile; buffer A=0.1% (v/v) TFA/H2O) at a flow rate of 1ml/min, and a wavelength of 230 nm.

Example 19 Compounds 20 and 21

Synthesis of keto-acids from compounds 18 and 19, the general procedureis the same as described in FIG. 4, except that compound 8 is replacedby compound 18 or 19, respectively.

Consequently, the general procedure can be represented by the followingscheme:

For Instance, Compound 20 is Prepared From Compound 18 as Follows:Compound 22.

Compound 18 (1 mmol, 1 eq) was dissolved in anhydrous DCM (10 mL). EDCIS(1 mmol, 1 eq) and DMAP (1 mmol, 1 eq) were successively added and themixture was stirred for 30 minutes. PPh₃CHCN (1 mmol, 1 eq) was addedand the resulting mixture was stirred at room temperature for 24 H.Reaction was quenched with 2% aqueous HCl solution. The organic layerwas then washed with saturated NaHCO₃ aqueous solution, dried over MgSO₄and concentrated under reduced pressure. The crude solid was purified byflash chromatography to give compound 22.

Compound 23.

In a flask connected to an oil bubbler, compound 22 (1 mmol, 1 eq) wasdissolved in 25 mL of anhydrous DCM:MeOH (7:3). The solution was thencooled to −78° C. and ozone was bobbled through it. Tha gas evolutionwas stopped during 5 minutes every 10 minutes. Despite the persistenceof the starting material, at the end of the reaction, the mixture waspurged with nitrogen. The solution was stirred for 1 h at roomtemperature and concentrated under reduced pressure§. The residue waspurified by flash chromatography to give the keto-ester 23.

Compound 20

Compound 23 (1 mmol, 1 eq) was dissolved in MeOH (7.2 ml) and 1N aqueousNaOH solution (2.1 mL, 2.1 eq.). The mixture was stirred for 1 h at roomtemperature, extracted with Et₂O and then acidified (pH=2) by the 5%aqueous HCl solution). The acidic aqueous layer was extracted with EtOAcand the combined organic phases was washed with brine and dried overMgSO4. The product was purified by precipitation (Hexane/DCM) to givecompound 20.

Example 20 Primary Cultures of Human Hepatocytes Represent an In VitroModel Suitable for Studying HCV Infection

1-1—Description of the Model of Study:

Interestingly, HCV was shown to be able to replicate in primary culturesof human hepatocytes infected in vitro, thanks to the activity of theviral RdRp (12).

The basic protocol was as follows: 1) human hepatocytes extracted fromliver lobectomies of HCV-negative patients were cultured in thelong-term culture system mentioned above; 2) cells were then infected invitro with an aliquot of serum from HCV-positive patients; 3) after afew days of culture (1 to 14 days), cellular RNA was extracted andanalyzed for HCV RNA (+) and (−) strands using a highly specific rtTh.The implemented RT-PCR method is as described in Sambrook and Russel,2001, Molecular cloning: a laboratory manual, 3^(rd) ed., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y.

A more detailed description of the experimental procedure used in thecontext of the present invention is given hereunder for one chemicalcompound (example 5). Of course, the procedure can differently be usedfor each compound described according to the present invention.

Three cultures of HCV-negative hepatic cells were used, named FT200,FT201, and FT203. These cells were extracted from metastasis of colictumors. (for the same tissue in three different patients).

Once in culture, hepatocytes were infected using a serum (S183) ofgenotype 3 from HCV-positive patients. Infection was performed in thepresence or the absence of a compound to be tested (here, compound ofexample 5), at concentrations of 0.1, 1, and 10 μM.

Cultures were maintained during 5 days with or without M107 treatment.

Cells were then washed and harvested.

RNA was extracted and analyzed using: first, rtTh RT-PCR and agarose gelelectrophoresis (control of the presence of the replicative strand); andsecond, quantitative RT-PCR using (a) the genomic strand of HCV, and (b)GAPDH RNA/GAPDH RNA as an internal control.

The results, illustrated in FIG. 5, were expressed as the ratio HCVRNA/GAPDH RNA, said ratio being normalized to 100 for cells infected butuntreated with compound 5.

1-2- Results:

As an example, a significant inhibitory effect of the compound ofexample 5 was obtained using 1 μM of said compound (see FIG. 5).

Thus, the compounds of the invention were found to have a stronginhibitory effect on the HCV in human hepatocytes.

Example 21

Operating Method for HCV NS3-4A Activity Assay:

Inhibition data were obtained on the full-length NS3/NS4A complex. Thetest has been described previously by Berdichevsky et al. (reference14). A summary of the NS3 protease activity assay is described bellow.

Principle:

We used the full-length NS4A-NS3 protease and a recombinantEGPF-NS5A/B-CBD fusion protein serving as a fluorescent substrate forthe determination of the compounds activity.

The fluorogenic substrate was built in three parts: a Green fluorescentprotein (EGFP), a peptide corresponding to the NS5A-NS5B junctions(ASEDWCCSMSYT) and a Cellulose Binding Domain (CBD). The NS5A/B is themore efficiently cleavage site for NS3 than at other cleavage site ofHCV polyprotein (references 15). After cleavage reaction ofEGFP-NS5A/B-CBD by NS3, the un-cleaved substrate and the C-terminaldomain of cleaved substrate are fixed on cellulose. Aftercentrifugation, the released free EGFP-NS5A was quantified byfluorometry.

Determination of the Activity:

The reaction was performed with reaction buffer 10× (500 mM Tris(HCl) pH7.5, 500 mM NaCl, 20% glycerol, 0.5% Tween, 1 mM DTT) at 37° C. Thefinal concentration of the enzyme was 50 nM and 100 nM for thefluorogenic EGFP-NS5A/B-CBD substrate, the final volume of reaction was150 μL.

The inhibitors were tested in triplicate in each experiment. Theinhibitor was pre-incubated with the NS3 enzyme (50 nM finalconcentration) for 5 minutes at 37° C. The substrate was added (100 nMfinal concentration) and incubated at 37° C. for 40 minutes. Then, thereaction mixture was transferred into an Eppendorf containing 5 mg ofpre-equilibrated cellulose with reaction buffer 1× and agitated at roomtemperature for 10 minutes followed by centrifugation for 5 min at 13000rpm. The supernatant (100 μL) containing liberated green fluorescentprotein was transferred to a black 96 well plate and data was collectedby a fluorometer (excitation λ=485 nm, and emission λ=538 nm). For eachblack 96 well plate the control of the activity and inhibition of NS3protease, fluorescence background noise and the measure of the totalfluorescence were performed. The fluorogenic substrate EGFP-NS5A/B-CBDat 100 nM final concentration was incubated with NS3 protease at 50 nMfinal concentration for the control of the NS3 activity. The inhibitorsAc-Asp-D-Gla-Leu-Ile-β-cyclohexyl-Ala-Cys-OH (CHAC), (references 16)PMSF and TPCK were used at various concentrations to validate theinhibition of NS3 protease reaction. The fluorescence background noisewas obtained with the fluorogenic substrate EGFP-NS5A/B-CBD at 100 nM,and the control of the maximum fluorescence was obtained with thefluorogenic substrate EGFP-NS5A/B-CBD at 100 nM without the step of thefixation on pre-equilibrated cellulose. HCV NS3 protease inhibition

NS3 Com- IC50 pounds R₁₀ R₁ R₃ R₅ (μM) 18 CO₂H CH₂—CH₃ CH(CH₃)₂ CH₃CO 3019 CO₂H CH₂—CH₃ CH(CH₃)₂ Ac-Val- 0.5 Hyp(OtBu)-  5 CO₂H CH₂—CF₃ CH₂—CH₂—Boc 150 CO₂H  6 CO₂H CH₂—CH₃ CH₂—CH₂— Boc 200 CO₂H CHAC — — — — 0.04PMSF — — — — 5000 TPCK — — — — 900

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Sathiyamoorthy, A. Golan-Goldhirsh, R. Tur-Kaspa, and I. Benhar. 2003. Anovel high throughput screening assay for HCV NS3 serine proteaseinhibitors. J. Virol. Methods 107:245-255.

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1-25. (canceled)
 26. A compound having the general structure (I) or (II)as follows:

R1 represents the side chain of an amino acid or an amino acidderivative, preferably of hydrophobic nature, an alkyl, alkenyl, oralkynyl group having from 1 to 10 carbon atoms, including CH2CH3 andCH2CF3; R2, identical or different, represents a hydrogen atom, an alkylgroup having from 1 to 10 carbon atoms, a hydroxyl function, an alkoxygroup, or an (C2-14)aryloxy group, —R2 may also represent a carbonylgroup (═O); R3, identical or different, represents the side chain of anamino acid or an amino acid derivative, preferably of hydrophobicnature, an alkyl, alkenyl, or alkynyl group having from 1 to 10 carbonatoms, or a, substituted or not, (C2-14)aryl or (C2-14)aralkyl group,the aryl moiety thereof being optionally interrupted by at least oneheteroatom; R4 represents a hydrogen atom, an alkyl, alkenyl, or alkynylgroup having from 1 to 10 carbon atoms; R5 represents a protecting groupfor the amine function R6 and R7 are the same or different and eachrepresents a hydrogen atom or an, linear, branched, or cyclic, alkyl,alkenyl, or alkynyl group having from 1 to 10 carbon atoms or a,substituted or not, (C2-14)aryl or (C2-14)aralkyl group, the aryl moietythereof being optionally interrupted with at least one heteroatom; R8and R9 are the same or different and each represents a hydrogen atom oran, linear, branched, or cyclic, alkyl, alkenyl, or alkynyl group havingfrom 1 to 10 carbon atoms or a, substituted or not, (C2-14)aryl or(C2-14)aralkyl group, the aryl moiety thereof being optionallyinterrupted with at least one heteroatom; R10 represents an aldehyde(—CHO), an acid group (—COOH), a sulfonic acid (—SO2OH), —COCOOH group,a radical selected in the group consisting of: —COR, —COOR, —CONRR′,—COCOOR, —SO2NRR′ (a sulfonamide group), —CONHCOR, —COCONRR′, —CONHSO2R,—CHOHCOR, —CHOHCOOR, —CHOHCON—RR′, R and R′, identical or different,represent an hydrogen atom, a hydroxyl radical, a linear, branched orcyclic alkyl, alkene or alkyne group having from 1 to 10 carbon atoms,an alkoxy group, an amine group or a, substituted or not, (C2-14)aryl,(C2-14)aralkyl, or (C2-14)aralkoxy group, the aryl moiety thereof beingoptionally interrupted with at least one heteroatom; n is 1 or 2; theirtautomers, optical and geometrical isomers, racemates, salts, hydratesand mixtures thereof.
 27. A compound according to claim 26, wherein thecompound corresponds to the following general formula (III):

wherein: R1, R2, R4, R5, R6, R7, R8, R9, R10 and n are as defined aboveand R11 represents a hydrogen atom, an alkyl group having from 1 to 10carbon atoms inclusive or a carboxy protecting group; their tautomers,optical and geometrical isomers, racemates, salts, hydrates and mixturesthereof.
 28. A compound according to claim 26, wherein the compoundcorresponds to the following general formula (III):

in which: R1 represents an alkyl group having from 1 to 10 carbon atomsinclusive or the side chain of an amino acid or an amino acidderivative, including CH2—CH3 and CH2CF3; R2 represents a hydroxylgroup, an alkoxy group having from 1 to 10 carbon atoms, or —R2 may alsorepresent a carbonyl group (═O); R4 represents a hydrogen atom; R5represents an amine protecting group; R6 and R7 are the same ordifferent and each represents a hydrogen atom, a linear or branchedalkyl group having from 1 to 10 carbon atoms or a cycloalkyl grouphaving from 1 to 10 carbon atoms, including a cyclohexyl derivative; R8and R9 are the same or different and each represents a hydrogen atom ora linear or branched alkyl group having from 1 to 10 carbon atomsinclusive; R10 represents an acid group, an ester group, an alkanoylgroup, a keto-acid, a keto-ester, a keto-amide or a α-hydroxy-ketoderivative; R11 represents a hydrogen atom, an alkyl group having from 1to 10 carbon atoms inclusive or a carboxy protecting group; and n is 1or 2; their tautomers, optical and geometrical isomers, racemates,salts, hydrates and mixtures thereof.
 29. A compound according to claim26, wherein the compound has the following formulae (Ia), (IIa) or(IIIa):

wherein R1, R2, R4, R5, R6, R7, R8, R9, R10 and n are as defined inclaim 1 and R11 represents a hydrogen atom, an alkyl group having from 1to 10 carbon atoms inclusive or a carboxy protecting group.
 30. Acompound according to claim 26, wherein the amino acid side chaincorresponds to any side chain of the naturally occurring (L form) orsynthesized (L or D form) aminoacids (in particular alpha-aminoacids andaminocyclopropanoic acid), or derivative thereof, optionallysubstituted.
 31. A compound according to claim 26, wherein the aminoacid side chain is selected in the group consisting of —CH3, —CH(CH3)2,—CH2—CH(CH3)2, —CH(CH3)C2H5, H, —CH2OH, —CH2CH3, —CH(OH)CH3, —CH2SH,—CH2CF3, —(CH2)2—S—CH3, —CH2CH2CF3, —CH3C2H5, —CH2C6H5, —CH2—C6H4(OH),—CH2CONH2, —(CH2)2CONH2, —CH2COOH, —(CH2)2COOH, —(CH2)4NH2,—(CH2)3NHC(NH2)2, —CH2CH═CH and C6H5.
 32. A compound according to claim26, wherein R5 stands for acetyl, benzyloxycarbonyl (Cbz) ort-butyloxycarbonyl (Boc) groups ; and/or R1 stands for —CH₂—CH₃,—CH₂—CF₃, —CH₂—CH₂—CF₃, —CH₂CHCH₂ or —CH₂−CHMe₂; and/or R2 stands fort-butyloxy; and/or R3 stands for —(CH₂)₂COOH, —CH(CH₃)₂, or—(CH₂)₂COOCH₃; and/or R10 is acid, —CHOHCOR, with R is OH or an alkoxygroup (preferably methoxy or ethoxy), keto-acid, keto-ester (preferably—COCOOMe, —COCOOEt or COCOOBn), keto-amide (preferably COCONHMe,COCONHEt or COCONHBn); and/or R4 is H; and/or R6 is H; and/or R7 is H;and/or R8 is H; and/or R9 is H; and/or R10 is H and/or n=1.
 33. Acompound according to claim 26, which is selected in the groupconsisting of:


34. A compound corresponding to the following formula (V):

wherein R2, R3, R4, R5, R6, R7, R8, and R9 are as defined in claim 26and R12 represents a hydrogen atom, an alkyl group (in particular,methyl, ethyl or t-butyl), alkenyl (allyl), an aralkyl (for instance,benzyl) or a cycloalkyl group; and n is 1 or 2; their tautomers, opticaland geometrical isomers, racemates, salts, hydrates and mixturesthereof.
 35. A compound according to claim 34, wherein it presents thefollowing formula (Va):


36. A compound according to claim 34, wherein it corresponds tocompounds of formula (V) wherein R6, R7, R8 and R9, independently fromeach other, represents a hydrogen atom, an alkyl, an alkoxy group, or acycloalkyl group, and preferably a hydrogen atom.
 37. A compoundaccording to claim 34, which is selected in the group consisting of:


38. A compound according to claim 34, useful as an active pharmaceuticalingredient, such as an antiviral agent (antiviral HCV agent).
 39. Apharmaceutical composition comprising at least one compound as definedin claim 26 and a pharmaceutically acceptable vehicle or support.
 40. Apharmaceutical composition according to claim 39, said compositionfurther comprising at least one immunomodulatory agent, other antiviralagent, other inhibitor of hepatitic C protease; inhibitor of othertargets in the HCV life cycle, or combinations thereof.
 41. Apharmaceutical composition according to claim 39, for treating a diseaserelated to an infection by a virus (preferably flavivirus, such asdengue virus, yellow fever virus, West Nile fever virus, or HCV),bacteria or pathogen dependent upon a serine protease for proliferation42. A pharmaceutical composition according to claim 39, for treating HCVinfection and the like.
 43. A pharmaceutical composition according toclaim 39, for treating hepatitis C virus infection and complicationsthereof, in particular chronic hepatitis, cirrhosis or hepatocellularcarcinoma and extrahepatic manifestation.
 44. A method for the treatmentof a disease associated with an infection by a virus (preferablyflavivirus, such as dengue virus, yellow fever virus, West Nile fevervirus or HCV), bacteria or pathogen dependent upon a serine protease forproliferation, by administering to subject in need of such treatment aneffective amount of at least one compound as defined in claim
 26. 45. Amethod for the treatment of a disease associated with HCV infection, byadministering to subject in need of such treatment an effective amountof at least one compound as defined in claim
 26. 46. A method ofevaluating the modulation properties of test compounds towards NS3serine protease, particularly HCV NS3 serine protease, said methodimplementing in vitro primary cultures of human hepatocytes andcompounds as defined in claims
 26. 47. A method for screening and/orcharacterizing compounds that present antiviral activity, in particularantiviral HCV activity, by implementing in vitro primary cultures ofhuman hepatocytes and compounds as defined in claim
 26. 48. A method forscreening and/or characterizing compounds that present antiviralactivity, said method comprising the following steps: a) contacting atest compound with the in vitro primary cultures of human hepatocytesdescribed herein in presence of HCV or active part thereof, and b)determining the antiviral activity of the test compound in comparisonwith the antiviral activity of one of the compounds as defined in claim26.
 49. A method to treat or prevent viral contamination of materials byimplementing at least one compound as defined in claim 26.